CN102876681A - Nucleic acid aptamer for targeted medicament carrier - Google Patents
Nucleic acid aptamer for targeted medicament carrier Download PDFInfo
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- CN102876681A CN102876681A CN2012103481076A CN201210348107A CN102876681A CN 102876681 A CN102876681 A CN 102876681A CN 2012103481076 A CN2012103481076 A CN 2012103481076A CN 201210348107 A CN201210348107 A CN 201210348107A CN 102876681 A CN102876681 A CN 102876681A
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Abstract
The invention discloses a nucleic acid aptamer for a targeted medicament carrier. The nucleic acid aptamer is a single stranded DNA (deoxyribonucleic acid) molecule showed by the sequence 1 in the sequence table. The experiments prove that the nucleic acid aptamer R13 provided by the invention is a section of DNA sequence obtained from the human lung cancer cell by adopting the cell selection method. The nucleic acid aptamer R13 is high affinity to the cell A549 and can enter the cell A549. The experiments in vitro prove that the nucleic acid aptamer R13 as the medicament-carrier carried light therapy agent TF70 greatly improves the cell A549 killing effect and has strong targeting performance.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of aptamer for target medicine carrier.
Background technology
Cancer is that a class causes human dead principal disease, and the annual whole world is approximately 7,000,000 because of the cancer mortality number.Along with the development of cytobiology and to the understanding of pathogenesis of cancer mechanism, the chemotherapy of cancer has obtained good development.Nearly 90 number of chemical drug developments are used for kill tumor cell and cancer therapy at present.But these medicines lack targeting specific, usually bring fatal side effect to the patient clinically.In a single day simultaneously, small molecules nucleic acid medicine has all been obtained reasonable result in anticancer experiment in vitro, but is applied in the body, will be decomposed by the enzyme in the human body; And external small molecules nucleic acid medicine is modified after, although can resist the cutting of enzyme, changed again the mechanism of action.Therefore need development to carry medicine attack tumour cell by target, can resist again the targeting vector of nuclease degradation.
Aptamer is the few nucleic acid molecule (ssDNA or ssRNA) of strand that the Fas lignand system evolution method by index concentration screens from the DNA/RNA library.It is folded into unique space structure by reactive forces such as base stacking in the molecule, hydrophobic, hydrogen bond and static, thereby high specific and high-affinity are identified its target molecules.The specificity that aptamer is combined with target molecules is similar to antibody with avidity, even stronger.Aptamer has lower molecular weight, non-immunogenicity, fast tissue permeability and good dynamic metabolism.Aptamer is in case confirm, the method for solid phase synthesis that just can be by automatization prepares, thereby has guaranteed that product can not there are differences between the different batches.Aptamer stable chemical nature and easily give its other function by chemically modified.
The more important thing is, compare with antibody, the target of aptamer is wider.The materials such as the somatomedin that some and disease are closely related, enzyme, acceptor and tumor markers can both become the target of aptamer.Aptamer for the vascular epidermis growth factor receptors is gone on the market by the FDA approval as the old moist macula lutea disease medicament for the treatment of at present; Also have simultaneously eight kinds of aptamer medicines to be in the different clinical investigation stages.Therefore carry the attention that medicine is attracting the investigator for the aptamer with the disease-related molecule as a kind of potential targeting vector.
Summary of the invention
An object of the present invention is to provide a kind of aptamer.
Aptamer provided by the invention, it is the single strand dna shown in the sequence 1 in the sequence table.
Above-mentioned aptamer also is the scope of protection of the invention as the application of pharmaceutical carrier.
Another object of the present invention provides a kind of pharmaceutical carrier.
Pharmaceutical carrier provided by the invention, its activeconstituents are above-mentioned aptamer.
In above-mentioned application or the pharmaceutical carrier, described medicine is following 1) or 2):
1) prevents and/or treats the medicine of tumour; Described tumour is specially adenocarcinoma of lung;
2) kill and wound and/or the medicine of inhibition tumor cell; Described tumour cell is specially lung adenocarcinoma cell; Described lung adenocarcinoma cell further is specially A549 clone.
The 3rd purpose of the present invention provides a kind of product.
Product provided by the invention is for being connected the product that obtains by above-mentioned aptamer with the target medicine; Described product is following 1) or 2):
1) prevents and/or treats the product of tumour;
2) kill and wound and/or the product of inhibition tumor cell.
In the said products, described target medicine is C70 three addition propanedioic acid oxalic acid (TF70); This compound is synthetic by the following method:
1) to 10mg C
70Middle adding 25ml toluene solution, dispersed with stirring is even, obtains C
70Toluene solution; In constant pressure funnel, add the 21ml toluene solution;
2) to C
70Passed into nitrogen in the toluene solution ten minutes, again to C
70Add 8.1 μ l bromo diethyl malonates in the toluene solution; Add 7.1 μ l DBU solution in the toluene solution in the constant pressure funnel, the jog funnel makes to be uniformly dispersed, and obtains containing toluene solution (bromo diethyl malonate: DBU=1:1, the DBU:C of DBU
70=6:1, the mol ratio of material);
3) continued to pass into nitrogen ten minutes, open constant pressure funnel, begin to drip the toluene solution that contains DBU, control solution added in one hour, continued reaction two hours, stopped reaction, filter with disposable filter, collect clear liquid in 50ml single port bottle, place the rear performance liquid chromatographic column of crossing that spends the night, moving phase is toluene, and flow velocity is 6ml/min.The sample introduction 2ml of elder generation observes retention time and is 40 minutes peak shape, determine to collect sample component and each sampling interval;
4) C behind the collection purifying
70Derivative, mass spectrometric detection is carried out in sampling.The rest part rotary evaporation is removed the toluene in the sample, adds 40ml ethanol.Ultra-sonic dispersion is attached to the C of bottle wall
70Derivative stirs and makes C
70Derivative is dispersed in the ethanolic soln as far as possible.Ethanolic soln (20mg NaOH, 1ml H with sodium hydroxide
2O, 9ml ethanol) hydrolysis C
70Derivative obtains corresponding richness and strangles acid derivative, filters with 0.2 μ m filter, solution is collected in the sterile chamber sealing for subsequent use, and 4 ℃ save backup, obtain TF70.
In the said products, above-mentioned aptamer be connected C70 three addition propanedioic acid oxalic acid and connect by amido linkage.
The said products is medicine, and wherein, described tumour is adenocarcinoma of lung; Described tumour cell is lung adenocarcinoma cell; Described lung adenocarcinoma cell is specially A549 clone.
Above-mentioned aptamer also is the scope of protection of the invention in the application that preparation prevents and/or treats in the product of tumour.
Above-mentioned aptamer preparation kill and wound and/or the product of inhibition tumor cell in application also be the scope of protection of the invention.
In the above-mentioned application, described tumour is adenocarcinoma of lung; Described tumour cell is lung adenocarcinoma cell; Described lung adenocarcinoma cell is specially A549 clone, and the said products is medicine.
Of the present invention experiment showed, the invention provides an aptamer R13, and it is the one section single strand dna that utilizes the cell screening method to obtain from human lung cancer cell A549's cell.It has very high avidity to the A549 cell, and, can also enter the A549 cell.In experiment in vitro, R13 carries light therapeutical agent TF70 as pharmaceutical carrier, greatly improves TF70 to the lethal effect of A549, and targeting is strong.
Description of drawings
Fig. 1 is that confocal detection aptamer R13 specificity characterizes
Fig. 2 is that flow cytometry is to the binding specificity sign of R13 and A549 cell
Fig. 3 is that the TF70-R13(amido linkage connects product) connection diagram
Fig. 4 is the cell survival rate result of medicine killer cell
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Part experiment material and reagent are as follows among the following embodiment:
Lung Adenocarcinoma A 549 Cell system is numbered CCL-185 available from ATCC;
Lung Adenocarcinoma A 549 Cell stable transfection EGFR-GFP(is provided by Chinese Academy of Sciences's Guangzhou biological medicine and health research institute from recombinant plasmid pEGFP-EGFR.Plasmid pEGFP is available from the Invitrogen of Ying Jun company), called after EGFR-A549 cell;
The cell culture fluid that is used for cultivation adenocarcinoma of lung A549 is the DMEM+10% foetal calf serum.
PBS damping fluid: pH is 7.4, is comprised of water and solute; Solute and concentration thereof are: 39mM NaH
2PO
4, 61mMNa
2HPO
4, 150mM NaCl;
Binding buffer liquid: pH is 7.4, is comprised of PBS damping fluid and solute; Solute and concentration thereof are: 1mM MgCl
2, 0.05%BSA, 0.2mg/ml yeast transfer RNA (tRNA) (Invitrogen company and catalog number 15401-011);
Lavation buffer solution: pH is 7.4, is comprised of PBS and solute; Solute and concentration thereof are: 1mM MgCl
2
FBS: foetal calf serum, available from the GIBCO(U.S.);
The acquisition of embodiment 1, aptamer R13
One, the acquisition of aptamer R13
1, the screening of aptamer R13
1), the pre-treatment in DNA library
After 5nmol DNA library (5 '-ACG CTC GGA TGC CAC TAC AG (40N) CTC ATG GAC GTG CTG GTG AC-3 ') dissolving, process through 94 ℃ of heating and cooled on ice, place 37 ℃ stand-by.
2), oppositely screening
Get 3 * 10
5Individual A549 cell adds pretreated DNA library, adds FBS, and (rotating speed 100rpm) hatched 1 hour in 37 ℃ of shaking tables, draws supernatant.
3), forward screening:
Get 3 * 10
5EGFR-A549 cell pearl adds the solution after instead sieving, and (rotating speed 100rpm) hatched 1 hour in 37 ℃ of shaking tables.Supernatant liquor is abandoned in suction; With PBS washed cell 2 times, scrape with cell again and cell is scraped off centrifuge washing 3 times (rotating speed 1000rpm), each 5 minutes.To add 500ulPBS in the EGFR-A549 cell, behind 95 ℃ of heating eluted dnas, tell centrifugal (12000rpm) to collect the template that supernatant liquor is used for pcr amplification.
2, screening and optimizing
In order to obtain high-affinity and specific aptamer, in the process of screening, progressively change screening time, just sieve and wash and wash number of times and increase screening pressure; With the confocal fluorescent microscope every the selection result of taking turns has been carried out preliminary investigation simultaneously.
3, PCR and ssDNA separate
The PCR reaction composition: 10 μ L, 10 * PCR buffered soln (100mM Tri s-HCl, pH 8.3; 15mMMgCl2), 3 μ L 1mM dNTPs, the upstream primer of 3 μ L25 μ M FITC marks (5 '-ACG CTC GGA TGC CAC TAC AG-3 ') and biotin labeled downstream primer (5 '-GTC ACCAGC ACG TCC ATG AG-3 '), 0.25 μ LTaq warm start polysaccharase, 5-15 μ L screening product and 69-79 μ L sterile purified water.Thermal cycle conditions: 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 30s.Before amplification, investigate different cyclic amplification products with agargel electrophoresis, choose suitable cycle index.
4, ssDNA separates
Utilize the coated affine absorption of dextran microballon of avidin, the sodium hydroxide of 0.2M separates double-stranded, and the ssDNA of FITC mark is separated with biotin labeled ssDNA.Again the ssDNA of FITC mark is crossed the NAP-5 pillar and carry out desalination, and collect filtrate between the 0.5-1.5mL.With the DNA concentration in the UV-vis spectrometry collection solution.DNA preserves or is used for the lower whorl screening in-20 ℃ after frozen cryodrying.
5, cloning and sequencing
After taking turns screening through 23, observation is with the instant sign of experiment, when fluorescence intensity no longer strengthens, getting last screening product is that template and unmarked upstream and downstream primer carry out pcr amplification according to above-mentioned PCR reaction conditions and step, the unmarked dna double chain product that obtains is operated according to pMD18-T Vector test kit handbook, then random sequencing.
6, sequential analysis
By analyzing the primary structure of ssDNA, then according to whether existing common conserved sequence that institute's calling sequence is classified in the stochastic sequence.
The result chooses 160 samples and checks order, and classify according to the DNA primary structure by the clone, obtains highly enriched fit sequence, and wherein a sequence has the specificity of height and can enter the A549 cell the A549 cell.Through sequence optimisation, final sequence is reduced and is: 5 ' TTTATGGGTGGGTGGGGGGTTTTT-3 ' (sequence 1); During sequence protection, with its similarity be that the sequence of 75%-90% all is similar sequence, the molecule of strand DAN shown in this sequence is aptamer R13.Above-mentioned aptamer R13 can artificial synthesized sequence 1.
Above-mentioned aptamer R13 can respectively with Cy3 and Cy5 mark (5 ' end of the fit R13 of the equal labeling nucleic acid of Cy3 and Cy5), obtain the fit R13 of Cy3 labeling nucleic acid and the fit R13 of Cy5 labeling nucleic acid.
Two, aptamer R13 specificity characterizes
1, confocal detection R13 specificity
The fit R13 of Cy3 labeling nucleic acid is added Lung Adenocarcinoma A 549 Cell nutrient solution (in DMEM+10% foetal calf serum cultivate obtain the A549 cell), and making the final concentration of the fit R13 of Cy3 labeling nucleic acid in total system is 2uM; Jointly hatch 18 hours (37 degree, 5% carbonic acid gas).
Do confocal detection (559nm excites) after hatching altogether, laser apparatus model: FV1000-IX81, Olympus, Japan), collect the microscope model: NA 0.40, UPLASPO, Olympus, result can find out as shown in Figure 1, find that aptamer R13 can enter the A549 cell, even can enter in the nucleus.
2, flow cytometry detects the binding specificity of R13 and A549 cell
Get Lung Adenocarcinoma A 549 Cell about 100,000, with 0.02%EDTA digestion, PBS washs secondary; Collecting cell, add respectively the fit R13 of Cy5 labeling nucleic acid of 200nM and the contrast dna sequence dna of 200nM Cy5 mark (5 '-AATTTTTTAATTATTTATATTA-3 ', Cy5 be marked at the contrast dna sequence dna 5 ' end), 37 the degree hatched 40 minutes; PBS washing once, with flow cytometer detect aptamer R13 and A549 cell in conjunction with situation.Do not add any dna fragmentation as contrast (A549 cell) take the A549 cell.
The result as shown in Figure 2,1 is the A549 cell, 2 is the combination of the control sequence of A549 cell and Cy5 mark, 3 is the combination of the R13 of A549 cell and Cy5 mark, can find out, compares with control sequence, R13 and A549 cell have very high avidity.
Can find out from above-mentioned experiment, aptamer R13 can enter the A549 cell and with its specific combination.
One, preparation TF70-R13(amido linkage connection product)
1, the preparation of TF70
TF70 is a kind of smooth therapeutical agent, is C
70Three addition propanedioic acid oxalic acid, its synthesis step is as follows:
1) to 10mg C
70Middle adding 25ml toluene solution, dispersed with stirring is even, obtains C
70Toluene solution; In constant pressure funnel, add the 21ml toluene solution;
2) to C
70Passed into nitrogen in the toluene solution ten minutes, again to C
70Add 8.1 μ l bromo diethyl malonates in the toluene solution; Add 7.1 μ l DBU(1,8-diazabicylo [5.4.0] 11 carbon-7-alkene in the toluene solution in the constant pressure funnel) solution, the jog funnel makes to be uniformly dispersed, and obtains containing toluene solution (bromo diethyl malonate: DBU=1:1, the DBU:C of DBU
70=6:1, the mol ratio of material);
3) continued to pass into nitrogen ten minutes, open constant pressure funnel, begin to drip the toluene solution that contains DBU, control solution added in one hour, continued reaction two hours, stopped reaction, filter with disposable filter, collect clear liquid in 50ml single port bottle, place the rear performance liquid chromatographic column of crossing that spends the night, moving phase is toluene, and flow velocity is 6ml/min.The sample introduction 2ml of elder generation observes retention time and is 40 minutes peak shape, determine to collect sample component and each sampling interval;
4) C behind the collection purifying
70Derivative, mass spectrometric detection is carried out in sampling.The rest part rotary evaporation is removed the toluene in the sample, adds 40ml ethanol.Ultra-sonic dispersion is attached to the C of bottle wall
70Derivative stirs and makes C
70Derivative is dispersed in the ethanolic soln as far as possible.Ethanolic soln (20mg NaOH, 1ml H with sodium hydroxide
2O, 9ml ethanol) hydrolysis C
70Derivative obtains corresponding richness and strangles acid derivative, filters with 0.2 μ m filter, solution is collected in the sterile chamber sealing for subsequent use, and 4 ℃ save backup, obtain TF70.
2, preparation TF70-R13(amido linkage connection product)
Above-mentioned 1 TF70 that obtains is connected with the aptamer R13 amido linkage that is obtained by embodiment 1 obtains TF70-R13; Connection diagram as shown in Figure 3, detailed process is as follows:
The TF70(0.5mg/ml that 0.1ml above-mentioned 1 is obtained) mix with the 2.0ml pure water, ultra-sonic dispersion ten minutes, (pH 5.8, and is 0.5M) for subsequent use, obtains TF70 solution to add 0.5ml MES damping fluid; The EDC aqueous solution (7mg/ml) and the Sulfo-NHS aqueous solution (13mg/ml) with fresh preparation adds in the TF70 solution again, the lower stir-activating of room temperature (25 ℃) adds the aptamer R13 that 5OD is obtained by embodiment 1 after 30 minutes, continue reaction and after 3.0 hours product is carried out ultrafiltration and dialysis (super filter tube molecular weight 10KD, dialysis tubing MW cut off 3500) to remove unnecessary reactant, obtains TF70-R13.
Two, R13 is as the targeting functional verification of target medicine carrier
The A549 cell is divided into following three groups of processing:
Control group (control): with 10
6The A549 cell in cell culture fluid, cultivated (37 degree, 5% carbonic acid gas) 3 hours;
TF70 group: with 10
6The A549 cell in the cell culture fluid that contains 10uM TF70, cultivated (37 degree, 5% carbonic acid gas) 3 hours;
TF70-R13 group: with 10
6The A549 cell in the cell culture fluid that contains 10uM TF70-R13, cultivated (37 degree, 5% carbonic acid gas) 3 hours;
Then be 20mW/cm with intensity all with above-mentioned 3 groups of cells
2Illumination, irradiation time is 5 minutes, 15 minutes, 30 minutes;
Shine after 30 minutes to count with cell counting count board and respectively organize cell quantity, utilize the CCK-8 method to come the reacting cells survival rate by the absorption value under the 450nm wavelength, experiment triplicate, results averaged.
The result can find out as shown in Figure 4:
The cell survival rate of control group (control) is 100%;
The cell survival rate of TF70 group is 85%;
The cell survival rate of TF70-R13 group is 8%;
Can find out from the above results, compare with independent adding TF70, illumination cultivation behind the adding TF70-R13, the mortality of A549 cell; Aptamer R13 is described as the target medicine carrier, has greatly improved the lethal effect of TF70 to the A549 cell.
Claims (10)
1. aptamer, it is the single strand dna shown in the sequence 1 in the sequence table.
2. aptamer claimed in claim 1 is as the application of pharmaceutical carrier.
3. pharmaceutical carrier, its activeconstituents is aptamer claimed in claim 1.
4. application according to claim 2 or pharmaceutical carrier claimed in claim 3 is characterized in that:
Described medicine is following 1) or 2):
1) prevents and/or treats the medicine of tumour; Described tumour is specially adenocarcinoma of lung;
2) kill and wound and/or the medicine of inhibition tumor cell; Described tumour cell is specially lung adenocarcinoma cell; Described lung adenocarcinoma cell further is specially A549 clone.
5. product is for being connected the product that obtains with aptamer claimed in claim 1 with the target medicine; Described product is following 1) or 2):
1) prevents and/or treats the product of tumour;
2) kill and wound and/or the product of inhibition tumor cell.
6. product according to claim 5, it is characterized in that: described target medicine is C70 three addition propanedioic acid oxalic acid;
Aptamer claimed in claim 1 be connected C70 three addition propanedioic acid oxalic acid and connect by amido linkage.
7. according to claim 5 or 6 described products, it is characterized in that: described product is medicine; Described tumour is adenocarcinoma of lung; Described tumour cell is lung adenocarcinoma cell; Described lung adenocarcinoma cell is specially A549 clone.
8. aptamer claimed in claim 1 prevents and/or treats application in the product of tumour in preparation.
Aptamer claimed in claim 1 preparation kill and wound and/or the product of inhibition tumor cell in application.
10. according to claim 8 or 9 described application, it is characterized in that: described tumour is adenocarcinoma of lung; Described tumour cell is lung adenocarcinoma cell; Described lung adenocarcinoma cell is specially A549 clone.
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CN109266653A (en) * | 2018-10-10 | 2019-01-25 | 中国科学院化学研究所 | A kind of reagent, the device and method of the capture of drug resistance heterogeneity circulating tumor cell and genetic analysis |
CN114436960A (en) * | 2021-12-10 | 2022-05-06 | 中国科学院化学研究所 | Photoactive aptamer conjugate drug for targeted therapy and preparation method and application thereof |
CN114634973A (en) * | 2022-03-11 | 2022-06-17 | 郑州大学 | Tumor exosome detection method based on aptamer recognition |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109266653A (en) * | 2018-10-10 | 2019-01-25 | 中国科学院化学研究所 | A kind of reagent, the device and method of the capture of drug resistance heterogeneity circulating tumor cell and genetic analysis |
CN114436960A (en) * | 2021-12-10 | 2022-05-06 | 中国科学院化学研究所 | Photoactive aptamer conjugate drug for targeted therapy and preparation method and application thereof |
CN114436960B (en) * | 2021-12-10 | 2024-03-26 | 中国科学院化学研究所 | Photoactive aptamer-coupled medicine for targeted therapy and preparation method and application thereof |
CN114634973A (en) * | 2022-03-11 | 2022-06-17 | 郑州大学 | Tumor exosome detection method based on aptamer recognition |
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